The overall goal of this project is to understand the molecular basis for the function of the cholesterol homeostasis signaling protein Scap. Integrated into the endoplasmic reticulum (ER) membrane of mammalian cells, Scap binds cholesterol and undergoes ligand-gated conformational changes that modulate the maturation of SREBP transcription factors. Scap is essential for cell survival, mediates the LDL-lowering activity of the statin drugs, and may be valuable as a drug target for cardiovascular and metabolic diseases such as hepatic steatosis. Despite the biological significance of Scap, remarkably little is known about the biophysical mechanism of its cholesterol binding and conformational changes. What is Scap's three- dimensional structure, and how is it changed by cholesterol binding to modulate Scap's interaction with downstream proteins(e.g. COPII) in the SREBP pathway? To understand how cholesterol binds to Scap, we will engineer a soluble form of Scap's cholesterol binding domain (CBD) that is amenable to biophysical characterization and structure determination. Comparison of cholesterol-free and bound CBD structures will provide our first molecular insights into sterol gating of Scap function, and will guide structure-based mutagenesis and functional assays. To discover how Scap links cholesterol binding to larger-scale structural changes that propagate across the membrane and ultimately modulate SREBP trafficking, we will identify an ortholog that is biochemically stable and tractable, and solve structures of the entire transmembrane region by single-particle cryoEM and X-ray crystallography. The combination of innovative protein engineering strategies, cryoEM, and lipid-mediated crystallography methods will overcome obstacles associated with membrane proteins and provide a detailed molecular picture of this physiologically essential sterol sensor. In the future we will use the knowledge and reagents developed in this project to help discover small-molecule Scap modulators.